Cancer PDF

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Cancer.ContentsIntro + nature of cancer.........................................................................................................................Bad luck vs preventable........................................................................................................................

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Cancer. Contents Intro + nature of cancer.........................................................................................................................1 Bad luck vs preventable.........................................................................................................................3 Cancer hallmarks/cell cycle/oncogenes.................................................................................................5 Tumour suppressor genes....................................................................................................................10 Viruses.................................................................................................................................................13 Mutagenesis/DNA repair.....................................................................................................................16 Metabolism/immortality.....................................................................................................................19 Common cancers.................................................................................................................................21 Chemotherapy.....................................................................................................................................25 Angiogenesis/metastasis.....................................................................................................................25

Intro + nature of cancer 1/3 people suffer from cancer in their lifetime. Rich countries have a higher incidence of cancer- this could be due to better diagnosis, people living longer due to control of infectious diseases, more sedentary lifestyle, more convenience foods. Cancer starts in single (monoclonal), normal cells and is progressive. Some cancers may originate from a polyclonal source where multiple cell types transform into cancer. The cell type must be one that stays in the body long enough to be able to accumulate multiple mutations and produces a progeny of transformed cells. Evidence for monoclonal cells- in female embryogenesis, random xinactivation occurs in the maternal or paternal chromosome, and varies between each cell type- but in tumour cells- the x inactivation is consistent (homozygote), suggesting that originated from only one cell type. Also, electrophoresis of plasma when normal, produces a smear- as there is a mix of immunoglobulins, but in multiple myeloma- there is a band of immunoglobulin- one type. Progression occurs from a normal into invasive carcinoma. The stages of cancer are normal cells  hyperplasia (increase in cell number)  dysplasia (abnormal cells)  in situ cancer  malignant. This can occur over a period of 20-30 years, when exposed to tobacco smoke. Neoplasia- new, uncontrolled growth of cells that is not under physiological control. This can be benign (adenoma) or malignant (carcinoma). Malignant cells are ones that have broken the basement membrane. Neoplastic tissue has a rosette appearance. Metaplasia- change in cell type. Anaplasia- loss of structural differentiation. 80% of all cancers start in epithelial cells, these are carcinomas. Squamous cell carcinoma- lines tissue. Adenocarcinomas- are in glandular or secretory epithelial tissue. Sarcomas- from mesenchymal cells e.g. connective tissue.

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Hematopoietic malignancies- from blood forming hematopoietic tissues. Neuroectodermal tumours- from cells in the CNS and PNS. The TNM cancer staging system is used worldwide. It is based on the size/extent of reach (T), spread to nearby lymph nodes (N), and presence of metastasis (M). The number next to each letter represents the severity/degree of spread.

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Bad luck vs preventable Some cancers are way more common than others- e.g. the large intestine has a 24 fold increased risk of cancer than the small intestine does. This could slightly be accounted for by the amount of time food spends in the system, but is mostly due to the amount of division occurring in the tissue. Tissues with a high cell turnover are more susceptible to cancers- e.g. stem cells that have unlimited differentiation potential, epithelial cells. However, there is not a lot of proliferation occurring in the brain but there are more cancer in incidences than in the small intestine. Divisions increase the chances of mutation and cancer. 2/3 of cancers can be explained by random (stochastic) mutations- including cell division. The research paper claimed that this was just ‘bad luck’, but still showed 1/3 of cancers had a strong environmental risk- measured by an extra risk score, excluding random mutations, e.g. a virus that causes liver cancer. However, environment has an effect on incidence of cancers, so a lot of them are preventable. If cancer was all caused genetically- incidence would be the same worldwide. People who move from one place to another, change their risk of cancer based on their new lifestyle choices. Chemical exposure can increase incidence of specific cancers. Other factors like high fat diet and smoking increase risk. The Ames test measures the mutagenicity of a compound- a safe strain of salmonella that requires histidine to grow (auxotroph) is exposed to the potential mutagen and is then put into culture without histidine. If salmonella grows, it shows that the bacteria has mutated as it does not require histidine to grow anymore, so is now a prototroph. Rat liver extract is also added to the mixture to assess the potential mutagenicity after metabolisation, in case the compound is not a direct mutagen. Salmonella is also grown on a plate without being exposed to a mutagen, to act as a control to see natural revertants.

Being mutagenic does not make a compound carcinogenic. This has to be determined by a long-term animal study, instead of bacteria- as they cannot develop cancer. Aflatoxin B1 is the most mutagenic and carcinogenic compound as it only requires a small amount to cause cancer. Benzidine is a polycyclic aromatic hydrocarbon (PAH) which is present in cigarette smoke and is also carcinogenic. MMS is an alkylating agent that is carcinogenic. Some compounds are carcinogenic not through damaging DNA and being mutagenic, but by acting as tumour promoters to increase proliferation. 3

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Cancer hallmarks/cell cycle/oncogenes Cancer cells show 8 hallmarks: 1. 2. 3. 4. 5. 6. 7. 8.

Sustain proliferation signalling Evade growth suppressors Resistant to cell death Immortal replication Induce angiogenesis Activate invasion and metastasis Avoid immune destruction Deregulate cell energetics

Enabling characteristics are required and favour the genetic changes of the cancer cell, these are: genome instability and inflammation, and tumour-promoting inflammation. Normal cell cycle- 4 stages- G1 (gap) , S (synthesis), G2 (gap), M (mitosis). Cells are in interphase most of the time. G1 phase senses the extracellular environment to see if it is appropriate to proliferatecontaining nutrients and growth factors. If inappropriate- the cycle will go back to G0 and become quiescent cells. Cells are responsive to growth factors and growth inhibitory factors at restriction points of the cycle. After passing the restriction point- cells are committed to divide, unless there is a disaster (metabolic, genetic, physical insults). Cells need growth medium- amino acids, glucose, fetal bovine serum (containing growth factors). Normal cells need to be stimulated by growth factors for proliferation to occur (mitogenesis), e.g. PGDF (platelet derived growth factors- platelets cause clotting and wound healing and proliferation), EGF (epidermal growth factors). Growth inhibitory factors inhibit proliferation e.g. TGF-B (tumour growth factor beta). Growth inhibitory factor TGF-B receptor is a serine/threonine receptor, not a tyrosine kinase receptor. It normally functions as a heterodimer. It antagonises/supresses tumour growth in most cases, but in progressed tumours it may not cause any response. Growth factors bind to growth factor receptors where signalling proteins activate transcription factors in the nucleus to cause a response (depending on which signalling cascade is activated)proliferation, differentiation, apoptosis. Growth factors are anchored in the membrane, with the ligand binding domain on the extracellular side and the tyrosine kinase domain intracellularly. They contain hydrophobic residues which allow them to move through the membrane lipids, and when they become in close proximity to another GF receptor- they dimerise and auto-phosphorylate to cause activation. EGF and HER (human epidermal) growth factor ligands can bind to receptors that dimerise and activate the tyrosine kinase activity and signalling cascade to the nucleus.

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Activation of a growth factor receptor. NSCLC (non small cell lung carcinoma) mutations are most often within the tyrosine kinase domain of a growth factor receptor, at the ATP binding site. In Glioblastoma, mutations are most often within the ligand binding domain. So different cancers (+ lifestyle choices) cause different most common locations of GFR mutations. Environmental factors can determine which types of mutations are more common e.g. 50% of non-smokers with lung cancer have EGFR mutations. The location of the mutation can help determine if the patient will be sensitive or resistant to a treatment drug. Cancer cells have genetic alterations in growth factor receptors, so they are activated more. 3 ways: 1) Mutations are in the tyrosine kinase domain, or through deletion of the ligand binding domain (need no ligand to become active) 2) Receptors are over-expressed, so that they often become in close proximity and autophosphorylate. 3) OR the cell expresses the growth factor receptor AND the ligand- so it will always be active (autocrine signalling).

Errors in growth factor receptors. Example of EGF going wrong: An avian retrovirus can induce cancer, and a mutation in a protooncogene- very similar to the EGF receptor- erbB, is responsible for this oncogenesis.

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Cyclin dependent kinases (serine/threonine kinases) regulate progression through the phases of the cell cycle. CDKs become activated when a cyclin binds to them, it becomes phosphorylated and undergoes a conformational change which promotes/activates its activity.

D-type cyclins (CDK4+CDK6) are the ones associated with the cell cycle, before the restriction point. Levels of cyclins fluctuate during the cell cycle (in a cyclic manner). Levels of cyclin D are upregulated during G1 from phosphorylation, and is downregulated with ubiquination and degradation. Mitogen stimulation increases cyclin D levels, in normal cells. CDK inhibitors/CKI’s (e.g. P16INK4- targets CDK4+CDK6, other complexes are inhibited by others e.g. P21) provide an extra level of control of cyclin-CDK complexes. They obstruct the ATP binding site to disable the kinase activity.

Mechanisms of CDK regulation- upregulated by cyclins, activating phosphorylation. Downregulated by inhibitory phosphorylation and CKI’s. pRb (retinoblastoma protein) is a converging point in the mitogen CDK pathway. pRb is a tumour suppressor gene, and for cells to pass the restriction point, it needs to be inactivated, through activation of CDK4/CDK6.

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Mitogenic signals (growth factors) allow the cell to progress out of the G0 stage, and activation of RB, through CDK4/6, allows the cell to pass the restriction point and be able to divide. In tumour cells, genetic alterations can change the signalling cascade through altering receptor tyrosine kinases (as mentioned earlier). Cancer mutations are often in CDKs- making them active, leading to permanent Rb inactivation and progression through the R point no matter what the circumstances are.

The cell cycle requirements and process of leaving G0. CDK4/6 are required to inactivate Rb, for DNA replication to occur. Molecular changes in cancers lead to deregulation of the cell cycle clock, in many possible ways e.g. inactivation of Rb, methylation of Rb promoter  inactivation, cyclin overexpression by many methods, defective degradation of cyclins, overexpression of CDKs .

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Tumour suppressor genes Cyclin D is often overexpressed in tumours. When proto-oncogenes acquire mutatations/overexpression/amplification, they become oncogenes. Growth factor receptors are oncogenes. Mutations in CKI result in increased activation of CDK . Tumour suppressor genes are inactive in cancer (both copies of the gene need to be supressed or there is still tumour suppression activity). Oncogenes are activated in cancer (only 1 copy is enough). Retinoblastoma is a cancer in often in children, the Rb protein is phosphorylated by CDK4 and CDK2. Disease can be unilateral or bilateral (1 eye or 2). The mutation can occur early in life and affect the eye stem cells, or born with the mutation in germline cells. Leukocoria is the most common signwhitening of the eye in flash photography. Disease is curable at the early stages. Inheriting retinoblastoma- need 2 copies of the Rb gene inactivated. Improvements in diagnosis allows children to survive and pass on their genes. Rb gene is located on chromosome 13, there is a deletion in retinoblastoma. Bilateral tumour cases are diagnosed earlier than children with unilateral retinoblastoma. This is because of the 2 hit theory: in unilateral cases- embryo was wildtype but during development 2 mutations occurred (2 tumour suppressor genes inactivated), in bilateral disease- children were born with 1 inactivated gene already (heterozygote germline mutation), 1 wildtype copy (mutates in embryogenesis)- only one hit is needed so bilateral disease presents earlier. People are predispositioned to cancer if they are born with 1 mutation in a tumour suppressor gene, as they only require one hit to affect both gene copies  cancer. This is the loss of heterozygocity (LOH). 40% of retinoblastomas are germinal/heritable and leads to bilateral disease. Incidence of other tumours is higher after bilateral retinoblastoma- they are predisposed as tumour suppressor Rb is inactivated. Treatment of the children early in life can also lead to development of mutations. Rb is a tumour suppressor gene that is phosphorylated by CDK4 and CDK6 in G phase of the cell cycle, and controls progress through the restriction point. Rb has many sites of phosphorylation so it can be hyper-phosphorylated. Rb binds to E2F to inhibit the transcription factor, but when Rb is phosphorylated, Rb is inactivated and E2F transcription factor can act. P53 is the most commonly mutated gene in human cancers. P53 is a transcription factor. People born with mutated p53 are predispositioned to early development of cancer. P53 is in chromosome 17, also the same chromosome as BRCA and HER. The ovary is the site with the most p53 mutations. Criteria for a tumour suppressor gene- mutations (inactivations) in the gene are found in cancer cases. Humans carrying germline mutations in the gene are more susceptible to cancer. Loss of the gene should lead to a cancer-prone phenotype in animal models. P53 (tumour suppressor) accumulates in tumour tissue, even if the mutation causes an inactivation of p53. Hotspots for mutation occur in the DNA binding domain of P53, and most mutations in p53 are (missense- codes for a different amino acid) point mutations. Li-Fraumeni syndrome occurs in people with germline p53 inactivation (1 copy)- predisposed to early cancer, and rare cancers. The mutation can skip generations and then re-appear. 10

When p53 is knocked out in mice- survival is low, and death occurs quickly, compared to having 1 or 2 wildtype copies of p53. 1 mutation still causes loss of heterozygosity and therefore lower survival, compared to 2 wildtype alleles. P53 acts as a tetramer (4 units), and the tetramerization domain is rarely mutated, compared to the DNA binding domain. P53 is activated by stress (e.g. DNA damage), and when phosphorylated during stress, it induces CKI and p53 tetramerises and travels to the nucleus to bind DNA to induce transcription of target genes (apoptosis, DNA repair, blocking of angiogenesis, cell cycle arrest).

Cancer cells have mutated P53 so they no longer go through apoptosis P53 can also cause apoptosis through activation of the death receptor (FAS) if FAS ligand binds. Also, inducing IGF binding protein (stopping the pro-survival InsulinGrowthFactor working). Also, inducing Bax- pores open in the mitochondrial membrane releases cytochrome c which promotes formation of the apoptosome to activate caspases- which kills the cell through apoptosis.

Rb is inactivated by phosphorylation, but p53 is activated by phosphorylation.

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Viruses Tumour development is a multi-step process- from accumulating multiple mutations- acquired from genetics, virus, diet, chemicals. Proto-oncogenes gain function and tumour suppressor genes lose function  cancer. Oncogenic viruses can induce these changes. Tumour cells are ‘transformed’- their lab features are: immortal (divide forever), no contact inhibition (division does not stop once cells contact each other- they do not form a monolayer)- and they can grow in soft agar, grow in low nutritional medium, forms tumors in mice with no T cells. They also have abnormal morphology, disrupted cytoskeleton, independent anchorage, contain new proteins. 1 in 5 of cancers are caused by an infectious agent in developing countries (Hep B/C- liver-HPVcervix/EBV/KSHV). This creates an opportunity to prophylactically treat people with vaccines, or treat the infection. HPV cervical cancer, throat and mouth. Non cancerous- warts. Untreated HIV indirectly is associated with cancer (KSHV- lymphomas, HPV- anogenital)- the immune system is important in preventing cancer. Viruses unintentionally cause cancer because they often infect dividing cells to be able to replicate themselves and grow using raw materials from the dividing cell- and allows persistence of the virus e.g. HPV. However, the drive for replication also increases the chance of cancer through oncogenes gaining function/loss of function in tumour suppressor genes. Rous sarcoma virus was the first oncogenic virus found- in chickens. Rous sarcoma carries a gene ‘vsarc’ (viral oncogene) which is related to ‘c-src’ (cellular proto-oncogene). V-src is a mutant- and has a selective advantage in the virus as it allows replication. Rous sarcoma virus has genes- GAG-POL-ENVSRC. Transducing viruses introduce new genes into a cell- they rapidly cause tumours to develop. The oncogene in the virus causes cancer in humans. A viral oncogene can transform cells by expressing the host cell related cellular oncogene, to drive the cell cycle. Viruses can convert the protooncogene into an oncogene in cells, as the virus oncogenes are over expressed from very efficient viral promoters and replicate more so have more mutations, whereas human proto-oncogene transcription is tightly controlled. Viruses/cancers caused by viruses: EBV- 95% of people are infected, causes glandular fever, but also burkitt’s lymphoma, hodgkins lymphoma, B cell lymphoma in AIDS patients. Burkitts lymphoma is a tumour in the jaw, and is common in central Africa- the cofactor to contribute to the condition is the malaria parasite. Hodgkins lymphoma- lymph node tumour. Nasopharyngeal carcinoma- throat and neck tumour. Patients have high antibodies for EBV, with ineffective T cell infiltrates. Occurs more often in a region of China, where people smoke cigarettes and eat salty fish- containing carcinogens. KSHV- lymph tumour. The morphology is spindle shaped due to the lack of contact inhibition. Primary effusion lymphoma- causes build up of fluids in body cavities, poor prognosis. 13

Multicentric castleman’s disease- disease of lymph nodes, often develops into cancer. Viruses activate the NFkB pathway to trigger anti-apoptotic, pro-inflammatory and proliferative signals to cause tumour. Signal transduction occurs to transmit a message from the outside of the cell where the TNF receptor is activated, phosphorylating iKb (inhibitor) ...